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BIOCHEM 3FS3 - Biochemistry of Food Science (All About Yogurt - Learning Module 7) This learning module will center around the biochemical and commercial importance of lactic-acid producing bacteria in the production of yogurt. The module has no set deadline, use it as you wish to develop to your own skills and knowledge. This course is inquiry based and self directed, and therefore relies on the student to drive their own learning forward. The following learning objectives are designed to fit individual student learning styles, so feel free to comment and modify them as necessary. 1. Read (and add discussion!) to the content summary presented below. 2. Answer the provided short-answer questions. Discussion with your peers is strongly encouraged, and additional research should be done to answer each question! 3. Come prepared for the in-class lab: "The Science of Yogurt Production", next Tuesday. What is yogurt? Yogurt is a milk product that is produced through bacterial fermentation, primarily with the species Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus. Lactose naturally present in milk is fermented by the bacteria to produce lactic acid. Continual production of lactic acid gradually lowers the pH of the milk culture, causing the formation of continuous acidic gel-like structures that gives yogurt its smooth consistency and sour taste. The name, yogurt, itself is a broad classification of several milk products that share the same preliminary step of bacterial fermentation. After fermentation, several techniques are used to modify the fermented milk product for different culinary applications, such as frozen yogurt, raita and tzatziki. Significance of yogurt fermentation Yogurt has been a staple food of human societies worldwide for thousands of years, and the global yogurt market is expected to exceed sixty-seven billion dollars by 2015.Dairy Products - A Global Strategic Business Report (2012). Global Industry Analysts Inc. Due to its adaptability, the yogurt recipe has been heavily modified throughout history by various cultural societies to suit their culinary and nutritional needs. Yogurt is rich in protein, calcium, riboflavin, folic acid, vitamin B6 and vitamin B12 Yale-New Haven Nutrition Advisor (March-10-05) Understanding yogurt. online http://web.archive.org/web/20080529005611/http://www.ynhh.com/online/nutrition/advisor/yogurt.html. and has a number of therapeutic applications such as protection against diarrhea, reducing lactose intolerance, prevention of urogenital infection, and the prevention of osteoporosis. Lourens-Hattingh, A., and Viljoen, B. C. (2001) Yogurt as probiotic carrier food. International Dairy Journal 11, 1–17 It is important to understand the fundamental biochemical processes involved in yogurt production to optimize industrial processing and ensure consumer safety. The mechanisms involved in yogurt production is an excellent example of fermentation, and can facilitate one’s understanding of energy metabolism in lactic acid bacteria. How is it made? Standardization Milk is first standardized to adjust the desired level of fat and protein content. This can be done by the addition of milk powders, whey protein concentrate, or milk protein concentrate. Stabilizers can also be added to the milk to improve the texture and appearance of the yogurt. Homogenization As fat in unprocessed milk normally settles at the surface, milk needs to be homogenized. Homogenization is the name given to any process that is used to mix two non-miscible solutions until evenly distributed. This is usually done by emulsifying fat into extremely small particles that can be dispersed within the aqueous component of milk. Several methods exist to homogenize milk, but one of the most common is to force unprocessed milk through small holes at high pressure, thus breaking fat globules into smaller particles and dispersing them within the solution.Trout, G. (1950) Homogenized milk : a review and guide. online http://library.wur.nl/WebQuery/clc/176536 (Accessed October 17, 2013). Pasteurization Homogenized milk is then pasteurized, which involves heating foods for a short period of time. One method, called the high temperature short time (HTST) method, involves heating milk to 72°C for 15 seconds before cooling to 4°C. HTST has been shown to kill 99.999% of bacteria found within milk.[1] Another method, ultra-high temperature (UHT) pasteurization further increases shelf-life by destroying all bacterial spores. However, UHT-treated milk leads to protein denaturation, contributing to an altered taste and a change in the viscosity of milk. Overall, pasteurization significantly increases the shelf-life of milk and is vital for the production of yogurt, as it prevents biological contamination. Addition of Bacterial Culture The next step involves inoculating milk with a bacterial culture and incubating at around 42°C until the product is firm. This change in viscosity occurs due to lactate production during anaerobic fermentation, which affects the structure and interaction of casein micelles in the milk (see “acidification” section below). The firmness and viscosity of yogurt is only one characteristic that plays into yogurt’s entire sensory evaluation, which also includes taste, acidity, smell, and appearance. Importance of bacterial metabolism to yogurt culture Both S. thermophilus and L. bulgaricus are facultative anaerobes, which have the ability to oxidize glucose using molecular oxygen if it is available. However, the conditions under which yogurt is made are generally anaerobic, requiring that both species shift their metabolism towards fermentation. In fact, both species grow best in anaerobic conditions, in part due to their inability to handle reactive oxygen species generated by aerobic metabolism. The role of starter cultures in yogurt production is primarily to acidify milk by converting lactose into lactate. The disaccharide lactose can be degraded to glucose and galactose, with the latter sugar entering glycolysis via the Leloir pathway (Figure 1A and B). In the Leloir pathway, a uridylyl pyrophosphate group is transferred to galactose from a molecule of UDP-glucose. The resulting UDP-galactose is epimerised to UDP-glucose and converted to glucose-1-phosphate, which rapidly equilibrates with glucose-6-phosphate (G6P) via catalysis by phosphoglucomutase. G6P is a product of the first step of glycolysis, and can therefore enter at the second step. Carbon shuttled into glycolysis ultimately finds itself in the form of pyruvate. The organism uses the free energy difference between glucose and pyruvate to synthesize ATP and reduce NAD+ to NADH (Figure 1C). In the absence of molecular oxygen as a final electron acceptor, the electron transport chain is unable to reoxidize NADH to NAD+, upon which glycolysis is dependent. To ensure the regeneration of NAD+ and the continual production of ATP from glycolysis, pyruvate is reduced to lactate using NADH as an electron donor (Figure 1D). As lactate builds, it diffuses outside of the organism. To maintain electroneutrality, protons diffuse out in equal proportions, thus acidifying the milk (Figure 1E). Acidification Yogurt develops a gel-like consistency when it is acidified by bacteria. This is due to the presence of isoelectric (pH 4.6) casein in its milk component. Casein is a protein that makes up 80% of proteins found in cow milk.[2] It is present in milk as a suspension of proteins and molecules called casein micelles, which is held together by colloidal calcium phosphate (CCP) molecules and hydrophobic interactions. The internal structure of casein micelles is disrupted by the solubilization of CCP in the acidic environment resulting from the production of lactic acid. The acidic environment also reduces the negative charge on the casein molecules, which decreases electrostatic repulsion and increases hydrophobic interactions between micelles. This, in turn, reduces the steric stability of the casein micelles, which allows the creation of clusters and aggregations of casein and thus the formation of a gel-matrix structure. Synergism between microorganisms S. thermophilus and L. delbrueckii subsp bulgaricus work synergistically to stimulate growth and acid production through a phenomenon known as proto-cooperation. Their combined efforts result in higher acidification rates, a lower final pH, increased bacterial populations, promoted aromatic compound production, and increased stability of final product. These two strains are able to work together by exchanging growth stimulants, such as folic acid and carbon dioxide, to compensate for the absence of various nutrients. In 2009, Herve-Jimenez et al. conducted a study to investigate the biochemical interactions between the two bacteria through post-genomic analyses of a co-culture with the two strains. Total RNA was extracted from the cultures at the 2.5 and 5.5 hour marks, and genome-wide analyses were performed on the extracted RNA using a commercial DNA microarray kit able to detect the genes present in the two strains. The pH and species-specific bacterial counts were also quantified every hour. It was discovered that when the two bacteria were cultivated together, the acidification of milk and bacteria counts were enhanced (see Figure 2). It was also found that in the coculture, there was an up-regulation of peptides, amino acid transporters, and amino acid biosynthetic pathways compared to the individual monocultures. To illustrate the symbiotic nature of the two strains, consider the up-regulation of Arg and BCAA biosynthetic pathways. Arg and BCAA are required for optimal growth in milk, particularly for protein synthesis. This biosynthetic pathway is present in S. thermophilus; however, it is not present in L. delbrueckii subsp bulgaricus. The latter bacterium benefits from being cocultured with the former by using its stores of Arg and BCAA. Surprisingly, S. thermophilus also benefits from the presence of L. delbrueckii subsp bulgaricus because depletion of its amino acid stores forces the strain to up-regulate amino acid biosynthesis in S. thermophilus, allowing for the enhanced growth of this strain. Acidity and growth of S. thermophilus cocultured with L. delbrueckii subsp. bulgaricus. (A) pH over time for monocultures of S. thermophilus (Δ) and L. delbrueckii subsp. bulgaricus (□) and for coculture (◆). (B) Bacteria counts over time for monocultures of S. thermophilus (Δ) and L. delbrueckii subsp. bulgaricus (□) and for coculture (■). Bacteria were harvested for proteomic and transcriptomic analysis at the early (E) (2.5 hour) and late (L) (5.5 hour) points of bacterial growth (arrows). Importance of Bacterial Metabolism to Yogurt Culture Both S. thermophilus and L. bulgaricus are facultative anaerobes, meaning that they have the ability to oxidize glucose using molecular oxygen if it is available.Zourari, A., Accolas, J. P., and Desmazeaud, M. J. (1992) Metabolism and biochemical characteristics of yogurt bacteria. A review. Le Lait 72, 1–34 However, the conditions under which yogurt is made are generally anaerobic, requiring that both species shift their metabolism towards fermentation. In fact , both species grow best in anaerobic conditions, in part due to their inability to handle reactive oxygen species generated by aerobic metabolism. Higuchi, M., Yamamoto, Y., and Kamio, Y. (2000) Molecular biology of oxygen tolerance in lactic acid bacteria: Functions of NADH oxidases and Dpr in oxidative stress. Journal of Bioscience and Bioengineering 90, 484–493 Condon, S. (1987) Responses of lactic acid bacteria to oxygen. FEMS Microbiology Letters 46, 269–280 The role of starter cultures in yogurt production is primarily to acidify milk by converting lactose into lactate.Lee, W. J., and Lucey, J. A. (2010) Formation and physical properties of yogurt. Asian-Aust. J. Anim. Sci 23, 1127–1136''' The disaccharide lactose can be degraded to glucose and galactose, with the latter sugar entering glycolysis via the Leloir pathway (Figure 1A and B). In the Leloir pathway, a uridylyl pyrophosphate group is transferred to galactose from a molecule of UDP-glucose. The resulting UDP-galactose is epimerised to UDP-glucose and converted to glucose-1-phosphate, which rapidly equilibrates with glucose-6-phosphate (G6P) via catalysis by phosphoglucomutase. G6P is a product of the first step of glycolysis, and can therefore enter at the second step. Carbon shuttled into glycolysis ultimately finds itself in the form of pyruvate. The organism uses the free energy difference between glucose and pyruvate to synthesize ATP and reduce NAD+ to NADH (Figure 1C). In the absence of molecular oxygen as a final electron acceptor, the electron transport chain is unable to reoxidize NADH to NAD+, upon which glycolysis is dependent. To ensure the regeneration of NAD+ and the continual production of ATP from glycolysis, pyruvate is reduced to lactate using NADH as an electron donor (Figure 1D). As lactate builds, it diffuses outside of the organism. To maintain electroneutrality, protons diffuse out in equal proportions, thus acidifying the milk (Figure 1E). Depending on the nature of the heat treatment, the milk undergoes gelation around pH 5.3, creating a firmer texture. '''THIS IS GOOD, JUST MAKE SURE YOU REFERENCE THE DIAGRAM MOAR, A MOAR DETAILED DIAGRAM WOULD ALSO HELP. WHY ARE WE YELLING?! Synergism between Microorganisms S. thermophiles and L. delbrueckii subsp bulgaricus work synergistically to stimulate growth and acid production through a phenomenon known as proto-cooperation.Zourari, A., Accolas, J. P., and Desmazeaud, M. J. (1992) Metabolism and biochemical characteristics of yogurt bacteria. A review. Le Lait 72, 1–34 Their combined efforts result in high acidication rates, a lower final pH, increased'' bacterial populations, promoted aromatic compound production and increased stability of final product, compared to monocultures. These two strains are able to work together by exchanging growth stimulants such as folic acid and carbon dioxide to supplement for the absence of various nutrients. In 2009, Herve-Jimenez ''et al. ''conducted a study to investigate the biochemical interactions between the two bacteria through post-genomic analyses of a coculture of the two strains.Herve-Jimenez, L., Guillouard, I., Guedon, E., Boudebbouze, S., Hols, P., Monnet, V., Maguin, E., and Rul, F. (2009) Postgenomic analysis of streptococcus thermophilus cocultivated in milk with Lactobacillus delbrueckii subsp. bulgaricus: involvement of nitrogen, purine, and iron metabolism. Appl. Environ. Microbiol. 75, 2062–2073 Total RNA was extracted from the cultures at the 2.5 and 5.5 hour marks, and genome-wide analyses were performed on the extracted RNA using a commercial DNA microarray kit able to detect the genes present in the two strains. The pH and species-specific bacterial counts were also quantified every hour. It was discovered that when the two bacteria were cultvated together, the acidification of milk and bacteria counts were enhanced (see Figure 2). It was also found that in coculture, there was an upregulation of peptides, amino acid transporters and amino acid biosynthetic pathways compared to coculture. Specifically, sulfur amino acid and galactose metabolism, the purine biosynthesis pathway, iron metabolism, and arginine (Arg) and Branched Chain Amino Acids (BCAA) biosynthetic pathways were stimulated. To illustrate the symbiotic nature of the two strains, consider the upregulation of Arg and BCAA biosynthetic pathways. Arg and BCAA are required for optimal growth in milk, particularly for protein synthesis. This biosynthetic pathway is present in ''S. thermophilus; ''however, it is not present in ''L. delbrueckii ''subsp ''bulgaricus. L. delbrueckii ''subsp ''bulgaricus ''evidently benefits from being in coculture with S. thermophilus by using its stores of Arg and BCAA. Surprisingly, ''S. thermophilus ''also benefits from the presence of L. delbrueckii subsp ''bulgaricus ''because depletion of its amino acid stores forces the strain to upregulate amino acid biosynthesis in S. thermophilus, allowing for the enhanced growth of this strain. How do we assess the quality of yogurt? The sensory evaluation of the final product should be prioritized over any other type of analysis.' 'Sensory evaluation has been receiving major support from the food industry recently as it is a cost-effective resource that can provide unique information that cannot be obtained from classical approaches. Sidel, J.L., and Stone, H. (1993) '''The role of sensory evaluation in the food industry. Food Quality and Preference. ''4,' 65--73 ''' '''''The food industry supports this evaluation as it allows them to create the highest quality product that will assume a large marketshare. To obtain this domninant marketshare, it is imperative that these companies use feedback from their eventual consumers using sensory evaluations. Sidel, J.L., and Stone, H. (1993) '''The role of sensory evaluation in the food industry. Food Quality and Preference. 4,''' 65--73 From a technical standpoint, researchers can only perform sensory evaluations, as there are no objective standards for testing the physical properties of the yogurt. Horiuchi, H., Inoue, N., Liu, E., Fukui, M., Sasaki, Y., and Sasaki, T. (2009) A method for manufacturing superior set yogurt under reduced oxygen conditions.'' J Diary Sci. ''92, 4112--4121 '' For example, in a 2009 study performed by Horiuchi et al, the researchers were attempting to study the effect of a LT-ROF (Low Temperature, Reduced Dissolved Oxygen Fermentation) production on the physical, sensory, properties, such as taste and consistency of the yogurt.Horiuchi, H., Inoue, N., Liu, E., Fukui, M., Sasaki, Y., and Sasaki, T. (2009) A method for manufacturing superior set yogurt under reduced oxygen conditions.'' J Diary Sci. ''92,' 4112--4121 Ultimately, the researchers were testing a new production method for yogurt, and if the yogurt was not pleasing to the general consumer's palate, the product would not sell, as consumer preference is mostly driven by physical properties such as flavour and consistency, and very little by chemical details such as the species of bacteria used or the exact pH of the yogurt. Ott, A., Hugi, A., Baumgartner, M., and Chaintreau, A. (2000) Sensory Investigation of Yogurt Flavor Perception: Mutual Influence of Volatiles and Acidity. J. Agric. Food Chem. ''48, 441--450 The commericial failure of the yogurt would mean that the the experimental production method is a failure regardless of what any data may say. The fact that the researchers used a large pool of consumers who regularly consume yogurt is a major positive, as the sensory evaluation from these individuals is from a panel of experts, which eliminates many potential biases. Thus, even though a sensory evaluation is subjective, it is the most important form of evaluation in any study regarding the production of foods. After all, yogurt is food, and who wants to eat bad tasting yogurt? Link Between Type II Diabetes and Dairy Consumption Recently, there has been studies that have suggested a relationship between diary consumption and incidence of type II diabetes. In 2005, Hyon et al. utilized a large prospective cohort study to demonstrate a link between the incidence of type II diabetes (T2D) and consumption of dairy products among men. Particularly, the authors found a significant reduction in risk of developing T2D within men who are in the top quintile of dairy consumption (RR=0.77).Choi HK, Willett WC, Stampfer MJ, Rimm E, and Hu FB (2005) Dairy consumption and risk of type 2 diabetes mellitus in men: A prospective study. Arch Intern Med 165, 997–1003 While its findings are of significance, one must be careful when drawing clinical conclusions from such data. The type of study is of significant concern, especially if used to influence clinical practice. Observational studies, while useful, do have significant limitations, such as the significant chance in selection bias due to the lack of randomization. It would be imprudent to derive clinical conclusions from the results of this study alone. However, given the impossibility of randomizing an exposure (dairy consumption), prospective observational data may be the best form of evidence available. Another area of concern is the lack of sample calculations to demonstrate that the study has sufficient power to make the conclusions that it has made. Instead, the authors merely stated that their study had a large sample size with no reference to statistical power or error. One other source of major error is that diagnosis of diabetes was achieved by self-reporting with a followup chart review. Under-diagnosis is of significant concern as screening for serum glucose is not always performed. To decrease bias, the data collectors and analysts should have been blinded to the patient’s exposure status. There is no evidence of this occurring. Finally, this study lacks external validity as only men between the ages of 40 and 75 with no prior history of the diseases were used as subjects, and thus any conclusions can only be generalized to this group. Whether if these findings apply to women or prevalent cases remains to be seen. One area in which the authors have succeeded is in the demonstration of a reasonable attempt at adjusting for confounding variables such as BMI, family history, and physical activity, which increases the internal validity of this study. Despite the study’s conclusion of an appreciable difference in relative risk between groups, the reader must not infer causality and use caution in allowing this study to influence clinical practice. The difference in RR, while detectable, is not sufficient to imply causality. Given the impracticality of an RCT, the demonstration of a dose-response relationship, or the proposal of a pathologically plausible mechanism, could be used to imply causality. These could be areas of further research to bolster the results of this prospective cohort study. Are there any pictures / figures that can go along with this section? Health Benefits of Probiotic Yogurt For health-conscious consumers, probiotic yogurt has been shown to have a stimulatory effect on cellular immunity, reduce cholesterol levels, and promotes intestinal tract health.Lourens-Hattingh, A., and Viljoen, B. C. (2001) Yogurt as probiotic carrier food. International Dairy Journal 11, 1–17 Probiotic yogurt uses microorganisms such as'' ''Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus ''GR-1 in the fermentation process. These probiotic bacteria play a role in promoting healthy flora growth by persisting in the intestinal tract and synthesizing organic acids that lower pH, thereby creating an intestinal environment less desirable for harmful bacteria, such as ''Salmonella spp. ''or various strains of ''E. coli. In particular, administration of probiotic bacteria has been shown to alleviate symptoms of lactose intolerance due to the auto-digesting properties of probiotic yogurt. In 2001, a study conducted by de Vrese et al. has shown that the autodigesting ability of probiotic yogurt can be partially attributed to the presence of β-galactosidase, a hydrolase enzyme that hydrolyzes the β-galactosidic bond present in lactose.Vrese, M. de, Stegelmann, A., Richter, B., Fenselau, S., Laue, C., and Schrezenmeir, J. (2001) Probiotics—compensation for lactase insufficiency. Am J Clin Nutr 73, 421s–429s Consumption of probiotic yogurt in lactose maldigesters has exhibited reduced diarrhea and flatulence. This auto-digesting feature of probiotic yogurt allows the consumption of yogurt in lactase-deficient individuals. References test